Antiviral drugs for smallpox are urgently needed because vaccination with "Dryvax" is unsafe for many people and because IL-4-containing poxviruses can subvert vaccine-induced protection. Cidofovir (CDV), an effective anti-poxvirus drug that must be given intravenously, has been improved by conjugating it with a hexadecyloxypropyl- (HDP) moiety. The resulting HDP-CDV is orally bioavailable and is now in final testing as an anti-smallpox drug. However, a second generation drug is needed because resistance to CDV can occur, and drugs frequently fail during the final stages of testing. Consequently, lipid esters of an adenine analog, (S)-9-[3-hydroxy-(2-phosphonomethoxy)propyl]-adenine ((S)-HPMPA), were produced. The advantages of HDP-(S)-HPMPA include: in vitro antiviral potency against poxviruses >60 times that of the current lead compound but with lower toxicity; oral activity in lethal models of cowpox and vaccinia in mice; protection from death even when given to mice 3 days after vaccinia infection; and a broad spectrum of antiviral activity against all dsDNA viruses and some retroviruses, including HIV-1. The alkoxyalkyl esters of (S)-HPMPA will be assessed and developed to attain four Milestones: (1) Lead optimization of the four most promising alkoxyalkyl esters of (S)-HPMPA by testing against poxviruses (including variola) in vitro, and by assessing oral activity in lethal challenge models with vaccinia, cowpox and ectromelia in mice, in vivo. (2) Evaluation of the best drug candidate by studies on oral pharmacokinetics, tissue distribution, metabolism, development of analytical methods for drug substance, stability testing, bioanalytical methods development, and 14 day GLP toxicology and histopathology in mice. (3) Preparation for IND submission, will focus on optimization of the chemical process for manufacturing, safety toxicology and genetic toxicology studies, and oral pharmacokinetics and a 14 day GLP toxicology and histopathology analysis in cynomolgus monkeys. (4) Evaluation of resistance to HDP-(S)-HPMPA using serially passaged vaccinia-WR and development of rapid nucleic acid testing for resistance mutations in the viral DNA polymerase. In conclusion, these studies are expected to yield an excellent drug candidate for the prevention and treatment of smallpox that is expected to be very close to Phase I clinical trials. HPMPA analogs are broad spectrum antivirals and other uses are anticipated which could make commercial development more likely.